PROJECT SUMMARY During the development of cancer, the incipient tumors exhibit signs of replication stress, including deregulation of origin firing, stalled forks, DSBs, and activation of p53. To what extent replication stress is a driving force in cancer can only be understood by further characterization of the pathways that stabilize and repair replication forks, allow restart for completion of replication, and induce a protective senescence or apoptotic state in precancerous cells. While HR (homologous recombination) proteins are involved in repair of stressed replication forks, their precise roles and regulation at forks are poorly understood compared to their function at DSBs. We will address these issues using the FA/BRCA (Fanconi anemia/Breast cancer) pathway of replication fork rescue as a focus. Recently, we demonstrated that DNA2 helicase/nuclease, plays a role in the FA/BRCA pathway. DNA2 is involved along with the MRN complex and BLM or WRN in resection of DSBs, giving rise to the 3? overhangs that induce the DNA damage stress response. These overhangs also recruit Rad51 to form filaments that participate in strand invasion during recombination and filament formation during stressed replication fork protection. Since DNA2 is also essential for DNA replication, we hypothesize that an important part of its HR/resection-related function is at genome destabilizing ssDNAs, gaps, DSBs or reversed forks arising during replication stress. Depletion of DNA2 suppresses chemosensitivity in FANCD2- deficient cells, and we will study the basis of this antagonism, which we propose is related to regulation of DNA2 resection by FA/BRCA pathway components. In Aim 1, we will use cytogenetics, genomics, and biochemistry to study the interplay of FANCD2 and DNA2 in choice of pathways (HR, NHEJ, and alt-NHEJ) that may compete for DNA ends or gaps arising during FA/BRCA repair of stalled replication forks. In Aim 2, we will further define the DNA2/FANCD2 interplay in replication fork protection and restart using iPOND and single DNA molecule analysis, highlighting novel analysis of FRA16D, a common fragile site that is unstable in FANCD2 deficient cells. In Aim 3, we describe an innovative, high resolution approach to studying the contribution of these factors to replication dynamics based on stalling replication at a chromosomal, site- specific protein/DNA replication block. A novel tool for our studies will be the DNA2 inhibitor we developed, which may also have therapeutic potential by sensitizing certain tumors to chemotherapies that cause replication stress.